S. M. Extraction

14/07/10

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Extraction of secondary metabolites for HPLC analysis

The biological material for the analysis

 Inoculate three Bells plates with agar plugs (4 – 8 mm) and incubate for 14 days at 20 oC in darkness.

 

Extraction:

  1. 5 plugs (ř = 7 mm) covering both new and old parts of the plate is made.

  2. Transfer the plugs to a 2 ml glass tube with teflon sealed lid.

  3. Add 1 ml of extraction solution and incubate for 1 to 10 hours at room temperature with 200 rpm horizontal shake (alternatively: use a ultrasound bath for 30 minutes).

  4. Transfer the supernatant to 1.5 ml Eppendorf tubes (do not store these as the extraction solution will attack the plastic).

  5. Evaporate the supernatant to complete dryness using a rotary evaporator or similar system.

  6. Resuspend the sample in 300 µl HPLC grade methanol (1 hour with 100 rpm).

  7. Spin the tubes at 20000 x g for 4 minutes.

  8. Transfer 200 µl of the supernatant to HPLC vials (can be used for storage).

  9. Analyse the sample by HPLC.

     

  • Materials needed:
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    Bells medium (pH = 5.1, N-source = 140 mg/L Urea, C-source = Maltose 20 g/L and no calcium carbonate) (ref. 1).

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    HPLC grade methanol (for resuspension) (4H gloves).

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    HPLC grade acetonitrile (as solvent for the HPLC) (nitrile gloves).

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    Extraction solution (methanol:dichloromethane:ethyl acetate in a 1:2:3 (v/v) ratio + 1 % (v/v) formic acid) (ref. 2) (H4 gloves).

     

     

         References.

         (1)             Bell, A.A. et al. Pest Management Science 59, 6-7 (2003): 736-747.

    (2)            Smedsgaard J. Journal of Chromatography A 760.2 (1997): 264-270.

    •  

    Safety considerations:

    Filamentous fungi are notorious for their production of toxic metabolites (with both acute and cumulative effects). For most of these the effects of long time exposure have not been examined and it is therefore a good idea to use gloves (latex or nitrile) when working directly with the mycelia or used growth media.

    Many of the chemicals used for this protocol is highly toxic (organic solvents) and great care should therefore be taken. Always use H4 gloves (or similar) when you are working with the “extraction solution” and “methanol”. All work should be performed in a fume hood to prevent inhalation of the chemicals. The use of gloves also protect against the fungal metabolites, which are very mobile in the used solvents, meaning that they will penetrate the skin easily.  

     

     
         

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    Dette sted blev sidst opdateret 14. July 2010